Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin to intracellular proteins, which is crucial for a wide range of biological processes. Polypeptides distinct from but related to ubiquitin - Ubls - can also be ligated to other proteins. Ubls have unique functions and, unlike ubiquitin, do not appear to target proteins for degradation by the proteasome. Defects in these pathways are being linked to a variety of disorders including cancer, immune system dysfunction, and Alzheimer's disease. Ubiquitin and Ubls are all synthesized as precursors, requiring enzymatic processing of C-terminal peptides or amino acids; furthermore, both ubiquitin and Ubl attachment to proteins is reversible. Specialized and highly conserved proteases are responsible for these processing reactions. The deubiquitinating enzyme (Dub) family in the yeast Saccharomyces cerevisiae has been the focus of the present grant. While trying to determine whether any members of this large group of proteases might process the Ubl called Smt3 or SUMO-1, a novel class of proteases unrelated to any Dub was discovered. These enzymes are highly conserved and play key roles in cell cycle progression and cell growth. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by Dubs and by the Smt3-cleaving enzymes. In this application, experiments that address two related areas are outlined. First, work on the yeast Dub called Doa4 will continue. Doa4 is a central enzyme of the ubiquitin system, and doa4 mutants are defective in an array of physiological pathways. To understand these defects, several mechanistic aspects of Doa4 action will be explored. In the second part of the application, experiments are proposed that examine the recently discovered yeast Smt3-specific proteases, Ulp1 and Smt4/Ulp2, using both molecular genetic and biochemical approaches. Specifically, they propose to: 1) isolate and characterize the ubiquitinated species that accumulate to high levels in mutant doa4 cells; 2) analyze the mechanistic connections between Doa4 and both the proteasome and the vacuolar-protein-sorting/endocytosis pathway; 3) identify the Smt3-protein conjugates from ulp1 and smt4 mutants and determine the functional consequences of Smt3 modification of these proteins; 4) characterize the regulation and specificity of Ulp1 by biochemical and molecular genetic approaches.